Noninvasive and invasive evaluation of cardiac dysfunction in experimental diabetes in rodents

Open Access

Original investigation

Noninvasive and invasive evaluation of cardiac dysfunction in

experimental diabetes in rodents

Rogério Wichi

_{, Christiane Malfitano}

_{, Kaleizu Rosa}

_{, Silvia B De}

Souza

_{, Vera Salemi}

_{, Cristiano Mostarda}

_{, Kátia De Angelis}

_{and Maria}

Claudia Irigoyen*

Address: 1_{Hypertension Unit, Heart Institute (INCOR), University of São Paulo, Medical School, São Paulo, Brazil,} 2_{São Judas Tadeu University,}

Human Movement Laboratory, São Paulo, Brazil, 3_{Nephrology Department, Federal University of São Paulo, São Paulo, Brazil,} 4_Experimental

Pathophysiology Program, University of São Paulo, Medical School, São Paulo, Brazil and 5_{Cardiomyopathies Unit, Heart Institute (INCOR),}

University of São Paulo, Medical School, São Paulo, Brazil

Email: Rogério Wichi - [email protected]; Christiane Malfitano - [email protected]; Kaleizu Rosa - [email protected]; Silvia B De Souza - [email protected]; Vera Salemi - [email protected]; Cristiano Mostarda - [email protected]; Kátia De Angelis - [email protected]; Maria Claudia Irigoyen* - [email protected]

* Corresponding author

Abstract

Background: Because cardiomyopathy is the leading cause of death in diabetic patients, the determination of myocardial function in diabetes mellitus is essential. In the present study, we provide an integrated approach, using noninvasive echocardiography and invasive hemodynamics to assess early changes in myocardial function of diabetic rats.

Methods: Diabetes was induced by streptozotocin injection (STZ, 50 mg/kg). After 30 days, echocardiography (noninvasive) at rest and invasive left ventricular (LV) cannulation at rest, during and after volume overload, were performed in diabetic (D, N = 7) and control rats (C, N = 7). The Student t test was performed to compare metabolic and echocardiographic differences between groups at 30 days. ANOVA was used to compare LV invasive measurements, followed by the Student-Newman-Keuls test. Differences were considered significant at P < 0.05 for all tests.

Results: Diabetes impaired LV systolic function expressed by reduced fractional shortening, ejection fraction, and velocity of circumferential fiber shortening compared with that in the control group. The diabetic LV diastolic dysfunction was evidenced by diminished E-waves and increased A-waves and isovolumic relaxation time. The myocardial performance index was greater in diabetic compared with control rats, indicating impairment in diastolic and systolic function. The LV systolic pressure was reduced and the LV end-diastolic pressure was increased at rest in diabetic rats. The volume overload increased LVEDP in both groups, while LVEDP remained increased after volume overload only in diabetic rats.

Conclusion: These results suggest that STZ-diabetes induces systolic and diastolic dysfunction at rest, and reduces the capacity for cardiac adjustment to volume overload. In addition, it was also demonstrated that rodent echocardiography can be a useful, clinically relevant tool for the study of initial diabetic cardiomyopathy manifestations in asymptomatic patients.

Published: 26 April 2007

Cardiovascular Diabetology 2007, 6:14 doi:10.1186/1475-2840-6-14

Received: 3 February 2007 Accepted: 26 April 2007 This article is available from: http://www.cardiab.com/content/6/1/14

© 2007 Wichi et al; licensee BioMed Central Ltd.

Background

Diabetes is a chronic metabolic disorder associated with secondary complications in the cardiovascular system and autonomic control in humans and animals [1-4]. Diabetic cardiomyopathy, a myopathic state independent of mac-rovascular complications, is the leading cause of death in diabetic patients [2]. Diabetic cardiomyopathy was impli-cated when diabetic patients at preclinical stages were found to exhibit a shortened left ventricular ejection time, a longer pre-ejection period, and elevated end-diastolic pressure [5]. However, the time-course of diabetic cardio-myopathy is still controversial, because reports have shown myocardial dysfunction at different times in the disease process.

Different methodologies have been used to investigate systolic and diastolic function in diabetic patients and rats. Assessment of cardiac function in experimental dia-betes has relied on ex vivo [3] or in vivo [6-8] techniques. The ex vivo technique requires sacrifice of the animals and is devoid of autonomic reflexes and ventricular-vascular coupling. Although LV cannulation is a well-established, precise invasive method, not devoid of autonomic reflexes and ventricular-vascular coupling, this technique is lim-ited because of the difficulty in keeping a catheter in the left ventricle throughout the long study period, and it is not without risk and does not allow assessment of the time course of cardiovascular changes. On the other hand, echocardiography has been used in several studies to demonstrate the time course of cardiovascular changes for different pathologies [9-12]. It is well established that clinically apparent diabetic cardiomyopathy may take sev-eral years to develop, but echocardiography can detect sig-nificant abnormalities early at the onset of symptomatic heart failure. Although several studies have used echocar-diography as one noninvasive methodology to identify cardiac dysfunction associated with diabetes mellitus, no previous studies have used one integrated approach, ie, echocardiography and in vivo hemodynamics, to evaluate cardiac function in an experimental diabetes model. So the purpose of the present study was characterization of early myocardial dysfunction performed at the same time by noninvasive echocardiography and invasive LV cathe-terization in STZ-induced diabetic rats.

Methods

Male Wistar rats (230 – 260 g) were obtained from the animal facilities at the University of São Paulo Medical School, São Paulo, Brazil. The rats received standard labo-ratory chow and water ad libitum. They were housed in individual cages in a temperature-controlled room (22°C) with a 12-h dark-light cycle. All procedures and protocols used were in accordance with the Guidelines for Ethical Care of Experimental Animals and were approved by the International Animal Care and Use Committee.

Experimental diabetic model

The rats were randomly assigned to 1 of 2 groups: control (C, N = 7), diabetic (D, N = 7). Animals were made dia-betic by a single injection of STZ (50 mg/Kg, iv; Sigma Chemical Co., St. Louis, MO, USA) dissolved in citrate buffer, pH 4.5. Food was withheld from the rats for 6 hours before STZ injection. Control rats received a pla-cebo (10 mM citrate buffer, pH 4,5) after a similar fasting period.

Twenty-four hour urine was collected 29 days after diabe-tes induction. Animals were placed in a metabolic cage and were allowed free access to food and water during the collection period. Urine was collected and centrifuged at 1,000 g for 10 minutes to remove particles, and the vol-ume was recorded. Urine samples were stored at -20°C for biochemistry analysis (Cobas Integra 700-Roche, Swiss).

Noninvasive evaluation of cardiac function

shortening (VCF) was measured following the formula (LVd-LVs)/(LVd × ET), where ET is the ejection time. The sample volume was then placed between the mitral valve and LV outflow tract so that the aortic valve closure line and the onset of mitral flow could be clearly identified. Isovolumic relaxation time (IVRT) was taken from aortic valve closure to the onset of mitral flow. Global cardiac function was evaluated by using the Myocardial Perform-ance Index (MPI), which is the ratio of total time spent in isovolumic activity (isovolumic contraction time and iso-volumic relaxation time) to the ejection time (ET). These Doppler time intervals were measured from the mitral

inflow and LV outflow time intervals. Interval "a_{", from}

the cessation to onset of mitral inflow, is equal to the sum of the isovolumic contraction time, ET and isovolumic

relaxation time. Ejection time "b_{" is derived from the}

duration of the LV outflow Doppler velocity profile. The

MPI was calculated with the formula (a-b_)/b_.

Invasive evaluation of cardiac function

LV function was measured also invasively in anesthetized rats (pentobarbital sodium, 40 mg/Kg). One catheter of PE-50 was inserted into the right carotid artery and advanced into the LV, and a second catheter of PE-50 was inserted into the jugular vein for saline and drug adminis-tration. Ventricular pressure signals were measured with a transducer and conditioner (Hewlett Packard 8805C, Waltham, MA) and digitally recorded (5 min) with a data acquisition system (WinDaq, 2-kHz, DATAQ, Springfield, OH). The recorded data were analyzed on a beat-to-beat basis to quantify changes in LV pressure. The following indices were obtained: heart rate (HR), LV systolic pres-sure (LVSP), LV end-diastolic prespres-sure (LVEDP), and maximum rate of LV pressure rise and fall (+dP/dt max and -dP/dt max). After LV pressure basal records, the LV func-tion was recorded during a volume overload protocol per-formed by an injection of saline (0.27 mL/min/kg) during 3 minutes adapted from Souza's study [13]. According to the volume overload protocol, the restoration of LV parameters was measured there for 3 minutes.

Data are reported as means ± SEM, and the Student t _test

was performed to compare metabolic and echocardio-graphic differences between different groups. Two-way ANOVA was used to compare LV invasive measurements, followed by the Student-Newman-Keuls test. Differences were considered significant at P < 0.05 for all tests.

Results

Rats treated with STZ not only developed hyperglycemia

(340 ± 7.6 vs. 87 ± 10 mg/dL, D vs. C, P _{< 0.0001) but also}

polydipsia (172 ± 6.5 vs. 40 ± 7 mL/24 h, D vs. C, P < 0.0001), polyuria (125 ± 4.4 vs. 9 ± 0.7 mL/24 h, D vs. C, P < 0.0001), glucosuria (12 ± 0.5 vs. 0.0026 ± 0.0003 mg/ dL, D vs. C, P < 0.0001), proteinuria (0.02 ± 0.002 vs. 0.01

± 0.0009 mg, D vs. C, P < 0.0001), and uremia (1.6 ± 0.09 vs. 0.27 ± 0.03 mg, D vs. C, P < 0.0001). Body weight was reduced in the diabetic group (270 ± 8 g) compared with weight in the control group (360 ± 11 g, P < 0.0001).

Noninvasive evaluation of cardiac function

Echocardiography 30 days after STZ demonstrated that LV internal dimension (cm) during diastole increased (0.73 ± 0.03 vs. 0.63 ± 0.03, D vs. C, P < 0.05), and the thickness of the interventricular septum and of the posterior wall during diastole decreased (0.101 ± 0.003 vs. 0.138 ± 0.005 and 0.1 ± 0.003 vs. 0.138 ± 0.005, D vs. C, P < 0.001, respectively) in diabetic rats in relation to control rats. RWT was significantly lower in diabetic (0.27 ± 0.016) than in control rats (0.40 ± 0.01, P < 0.05). LV systolic function, as expressed by fractional shortening (%) (34 ± 3.7 vs. 39 ± 3.7, D vs. C, P < 0.05), ejection frac-tion (%) (69 ± 0.02 vs. 75.4 ± 0.015, D vs. C, P < 0.05) and VCF (circ/sec) (0.003 ± 0.0002 vs. 0.004 ± 0.0003, D vs. C, P < 0.01) was reduced in diabetic group compared with that in the control group. LV diastolic function was observed after 30 days of STZ-induced diabetes, as expressed by reduced E-wave (m/s) (0.44 ± 0.036 vs 0.53 ± 0.04, D vs. C, P < 0.05) and increased A-wave (m/s) (0.4 ± 0.04 vs. 0.33 ± 0.02, D vs. C, P < 0.05) in diabetic ani-mals when compared with control aniani-mals. The diabetic group had a reduced E/A ratio (1.33 ± 0.09) compared with the control group (1.62 ± 0.07, P < 0.05). Decelera-tion time of E-wave (EDT, ms) (39 ± 1.3 vs. 30 ± 1.7, D vs. C, P < 0.0001) and IVRT (ms) (40.6 ± 1.65 vs. 31.6 ± 1.5, D vs. C, P < 0.05) were increased in the diabetic group compared with the control group, reflecting early diastolic dysfunction by slow relaxation. Statistical comparison of normalized EDT and IVRT by HR during echocardiogra-phy corroborated differences between groups observed using their absolute values. MPI was greater in the diabetic group (0.41 ± 0.014) compared with that in the control group (0.29 ± 0.054, P < 0.0001).

Invasive evaluation of cardiac function

During and after volume overload, the differences in LV function between groups remained the same as in the baseline period. However, the LVEDP increased in both groups during and after volume overload compared with their respective baseline values. Only diabetic rats showed an increase in LVEDP values in the post volume overload period in relation to LVEDP values during the volume overload period (Table 1).

Discussion

The present study provides an integrated approach to the assessment of diabetic cardiac function in rats, using both noninvasive echocardiography and invasive hemody-namic evaluation; it provides important new insights into the pathophysiology of diabetic cardiomyopathy. The present data confirm our preliminary findings that STZ diabetes induces hyperglycemia, weight loss, and brady-cardia. However, the major finding of this study is that rats with 30 days of STZ-induced-diabetes had impair-ment in cardiac structure and function as evidenced non-invasively by echocardiography and non-invasively by LV catheterization, and also that this LV dysfunction was exacerbated by volume overload.

Noninvasive evaluation of cardiac function

Echocardiographic measurements of cardiac function and structure in the present experiments demonstrated both LV systolic and diastolic dysfunction in 30-day diabetic rats. In fact, several investigators have shown abnormali-ties in LV systolic function, diastolic function, and com-plaints in humans [10-12] and rats [9,14-16], by using a noninvasive method of evaluation. Joffe et al [7] observed diabetic cardiomyopathy, characterized by LV systolic and

diastolic dysfunction, after two and a half months of STZ in rats. Akula et al [9] in a recent study used echocardiog-raphy to examine LV function in STZ rats over a definite course of time (2, 4, 8, and 12 weeks). They concluded that LV systolic and diastolic dysfunction was fully visible at 12 weeks of diabetes by this method, and that echocar-diography is useful in diagnosing cardiac abnormalities in diabetic rats without the need for invasive histopatholog-ical procedures. In contrast with these authors, we observed systolic and diastolic dysfunction after 30 days of STZ-induced diabetes documented by reduced ejection fraction, fractional shortening, and E-wave and increased A-wave and EDT, as well as by reduced VCF and increased IVRT observed in diabetic animals compared with con-trols. Corroborating our data, Yu et al [17] observed sig-nificant deficits in myocardial morphology and functionality by using magnetic resonance imaging at 4 weeks of diabetes in STZ-induced diabetic mice. In another study, Mihm et al [14] showed reductions in HR and EDT after 3 days of STZ-induced diabetes, and these reductions progressed throughout the 56-day period. Fur-thermore, these authors observed that systolic dysfunc-tion was detected only after 35 days of study.

LV cavity dilatation accompanied by no changes in wall thickness has been observed previously after 35 and 75 days [7,14] and 12 weeks [9] of diabetes. Joffe et al [7] demonstrated not only LV cavity dilatation, but also increased LV mass after 75 days of STZ induction in rats, suggesting evidence of cardiomyopathy characterized by eccentric hypertrophy. In contrast, in the present study, we observed LV cavity dilatation accompanied by decreased thickness of the LV posterior wall and

interven-Table 1: Left ventricular function at baseline, during, and after volume overload in the control and diabetic groups.

Parameter Group (n = 7) BASELINE VOLUME OVERLOAD POST VOLUME OVERLOAD

1 min 2 min 3 min 1 min 2 min 3 min

LVSP C 136 ± 4.7 125 ± 6.5 135 ± 7 134 ± 6 139 ± 7.8 134 ± 8.8 138 ± 6

(mm Hg) D 114 ± 6* 116 ± 5.8* 112 ± 5 * 114 ± 6 * 107 ± 10 * 102 ± 4 * 107 ± 10*

LVEDP C 4.6 ± 0.3 7.8 ± 0.6 8.0 ± 0.6 9.1 ± 0.56 11 ± 1.3 9 ± 1.1 9.4 ± 1.1 (mm Hg) D 7.4 ± 0.2* 9.9 ± 0.5*# 12.3 ± 0.55*# 11.5 ± 0.72*# 17 ± 1.3 *#† 16 ± 0.7 *#† 15 ± 1.1 *#†

HR C 350 ± 14 327 ± 10 333 ± 11.5 340 ± 7.5 352 ± 5 360 ± 6 360 ± 6

(beats/min) D 307 ± 8.5* 300 ± 6 * 300 ± 7* 302 ± 7* 297 ± 1.5* 298 ± 2* 294 ± 3.5 *

+dP/dt max C 9229 ± 463 8052 ± 741 8699 ± 653 8595 ± 457 8858 ± 504 9273 ± 569 9264 ± 603 (mm Hg/sec) D 6566 ± 609 * 5861 ± 584 * 5760 ± 770 * 6087 ± 1017* 5320 ± 1287 * 5844 ± 1217* 5712 ± 1247*

-dP/dt max C -6845 ± 379 -6446 ± 576 -6543 ± 600 -6139 ± 514 -6672 ± 646 -6320 ± 858 -6272 ± 707 (mm Hg/sec) D -4746 ± 589* -4804 ± 580* -4678 ± 580* -4152 ± 694* -3706 ± 614 * -3557 ± 561* -3246 ± 653*

tricular septum after 30 days of diabetes, suggesting a reduction in LV mass. Similarly, Dobrzynki et al [18] observed reduced heart weight and LV mass associated with cardiac and renal function damage after 21 days of STZ-induced diabetes in rats. In addition, our study is the first to describe the RWT reduction in diabetic rats, indi-cating a probable reduction in LV mass, as previously demonstrated [19].

MPI is a simple and nongeometric index that combines systolic and diastolic functions independently of heart rate, not requiring frequency based normalization [20]. The importance of MPI in this study is that it is an HR-independent measure unlike transmitral flow velocities. MPI also has not been used previously to investigate car-diomyopathy in diabetic rats. In our evaluations, MPI was

increased in diabetic rats, suggesting global cardiac dys-function in these animals. This result appears to correlate well with invasive measurements of systolic and diastolic measurements in humans [20] and animals [21]. Recently, MPI was validated in mice, and is strongly corre-lated with invasive measurements of the LV dP/dt max [21]. MPI has a prognostic value after myocardial infarc-tion [22], as well as in adults with amyloidosis [23] and dilated cardiomyopathy [24], and is not affected by mitral regurgitation [25].

Invasive evaluation of cardiac function

Dent et al [16] demonstrated that differences in diastolic function might be noninvasively quantified in diabetic hearts; however, these authors recognized that the lack of in vivo hemodynamic data was one limitation of their

Left ventricle cavity (a), E-wave (EW, b), A-wave (AW, b), original record of basal left ventricle pressure (c), and +dP/dt max and -dP/dt max of left ventricle pressure (d) in control and diabetic rats

Figure 1

Left ventricle cavity (a), E-wave (EW, b), A-wave (AW, b), original record of basal left ventricle pressure (c), and +dP/dt max and -dP/dt max of left ventricle pressure (d) in control and diabetic rats.

E

A

EW

Control

Diabetic

160

mm

H

g

/sec

mm

H

g

-10

-10 000 10 000

a

b

c

d

AW EW

study. In our experiments, direct measurements of cardiac function corroborate diastolic and systolic LV function impairment observed after 30-day-induced diabetes by the echocardiographic approach. In vivo LV function evi-denced reduced LVSP and +dP/dt max, reflecting a systolic dysfunction, increased LVEDP and attenuated -dP/dt max, showing diastolic dysfunction. These data associated with the impairment in MPI reported in the diabetic group in the present study corroborate the positive correlation between these ventricular indexes, as previously shown in mice [21]. In fact, similar alterations in LVEDP, contractil-ity, bradycardia, reduced cardiac output, and renal dam-age were evidenced after 21 days of STZ in rats. Reduction in HR in diabetic rats has been attributed to changes in sinoatrial node [1,3,26], although functional alterations in the cholinergic mechanism cannot be excluded as a causal factor. Joffe et al [7] showed the in vivo reduced peak of LV systolic pressure and increased LVEDP, as well as in vitro attenuation of LV +dP/dt max and -dP/dt max after 75 days of STZ-induced diabetes in rats. Another study in which in vivo LV cannulation was performed showed a decrease in LV +dP/dt max and -dP/dt max after 15 days of STZ injection in rats [8]. Impairment in cardio-vascular function in mice was also observed in diabetic-infarcted mice at the same time point [27]. In a previous study by our group, we reported that the isolated hearts of 11-week STZ-induced diabetic rats did not have differ-ences in LV isovolumetric systolic pressure, but had reduced contractility compared with isolated hearts in control rats [3].

In addition, our results show impairment in cardiac responses during and after volume overload in STZ-induced rats. A similar increase in LVEDP observed in con-trol rats in the present experiments was previously dem-onstrated in the early stages of volume overload induced by A-V fistula in control rats [28]. However, in contrast with that observed in control rats, the high values of recorded LVEDP in STZ-induced rats remained elevated after volume overload, which may be explained by cardiac changes produced by STZ. Despite the fact that LV cavity dilatation allows major blood compliance, the reduced fractional shortening, ejection fraction, and VCF observed in STZ-induced rats are probably associated with the increase in LVEDP during and after volume overload, because the volume of blood can not be completely ejected.

Studies that have examined both systolic and diastolic dysfunction in diabetes suggest that the latter is more sus-ceptible to preclinical changes. Diastolic dysfunction is not just a defect in active relaxation, but also in passive stiffness of the left ventricle [29]. The echocardiographic evidence of subclinical contractile dysfunction and diasto-lic filling abnormalities are predictive of subsequent

chronic heart failure [30]. Patients with diastolic heart failure have an increased mortality of 5–8% compared with the control group [31]. Systolic dysfunction occurs late, often when patients have already developed signifi-cant diastolic dysfunction. However, the prognosis in patients with systolic dysfunction is an annual mortality of 15–20%, greater than mortality in patients with diasto-lic dysfunction [31]. Thus, the early detection of diastodiasto-lic and systolic dysfunction can prevent worsening of this condition.

Conclusion

Using the STZ model of diabetes, we have demonstrated that rodent echocardiography can be useful, because sen-sitive changes in systolic and diastolic performance were detected and markedly confirmed by in vivo direct LV measurements. Changes reported in the present experi-ments in cardiac performance are highly predictive of clin-ical findings, and further illustrate the value of this model of diabetic cardiomyopathy.

Abbreviations

LV: left ventricle

STZ: streptozotocin

AP: arterial pressure

HR: heart rate

LVd: left ventricle end-diastolic diameter

LVs: left ventricle end-systolic diameter

PWd: end-diastolic posterior wall thickness

IVSd: end-diastolic interventricular septum thickness

RWT: relative wall thickness

E: maximal early diastolic peak velocity

A: late peak velocity

VCF: velocity of circumferential fiber shortening

ET: ejection time

IVRT: isovolumic relaxation time

MPI: myocardial performance index

LVSP: left ventricular systolic pressure

+dP/dt max: maximum positive values of first derivative of left ventricular pressure over time

-dP/dt max: maximum negative values of first derivative of left ventricular pressure over time

EDT: deceleration time of E-wave

Competing interests

The author(s) declare that they have no competing inter-ests.

Authors' contributions

All authors have equally contributed to the conception and drafting of the manuscript.

Acknowledgements

The authors acknowledge the financial support from FAPESP (01/00009-0; 01/07632-5; 04/04283-8) and CNPq.

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